The Role of the Kharon Complex in Leishmania Virulence

卡伦复合体在利什曼原虫毒力中的作用

• 批准号: 9101973
• 负责人: Scott M Landfear
• 金额: $ 19.25万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9101973
• 关键词: Address; African; Attention; Biochemical; Biotin; Blood Circulation; Ca(2+)-Transporting ATPase; Cells; Complex; Culture Media; Cytosol; Data; Defect; Disease; Electron Microscopy; Environment; Flagella; Fluorescence; Fluorescence Microscopy; Genes; Glucose Transporter; Health; Human; Immunofluorescence Microscopy; Impairment; In Vitro; Infection; Insecta; Integral Membrane Protein; Invaded; Label; Laboratories; Leishmania; Leishmania mexicana; Lesion; Life; Location; Mediating; Mediator of activation protein; Membrane; Membrane Proteins; Microscopic; Molecular Weight; Mus; Organelles; Parasites; Phagolysosome; Play; Property; Proteins; Protozoa; Publishing; Resolution; Role; Signal Transduction; Staging; Surface; Synapses; Testing; Trypanosoma; Trypanosoma brucei brucei; Vesicle; Virulence; base; cell motility; extracellular; macrophage; member; mutant; novel; parasitism; permease; protein complex; research study; synaptogenesis; trafficking

 DESCRIPTION (provided by applicant): Relevance: Parasitic protozoa such as Leishmania and Trypanosoma infect millions of people worldwide and cause devastating and fatal diseases. This application will elucidate an important mechanism whereby these parasites successfully invade human cells and cause disease. Summary. Recent discoveries have revealed that the whip-like flagellum that mediates motility of these single-cell parasites plays a critical role in he disease causing stages, the amastigote of Leishmania and the bloodstream form African trypanosomes. Specifically, our laboratory has discovered a novel protein, KHARON1, that is involved in targeting integral membrane proteins to the flagellar membrane in Leishmania mexicana. Importantly, KHARON1 is essential for disease-causing amastigotes of L. mexicana to survive inside the phagolysosomal vesicles of human macrophages. Although Δkharon1 null mutants replicate as axenic amastigotes in culture medium, once they enter host macrophages they do not replicate but rather die inside these host cells. These results suggest a role for KHARON1 specifically in survival of amastigotes inside the macrophage phagolysosome. Recent experiments also indicate that Δkharon1null mutants are avirulent following infection of Balb/C mice. Other preliminary data establish that KHARON1 is part of a high molecular weight multi-protein complex designated the KHARON Complex. The overall objective of this application is to dissect the critical function of the KHARON Complex in disease causing amastigotes and to thus illuminate the role of the amastigote flagellum in parasite virulence. The KHARON1 protein, which localizes to the base of the flagellar axoneme in insect stage promastigotes, will be localized in amastigotes by high-resolution fluorescence microscopy and electron microscopy. The ability of KHARON1 to mediate trafficking of a membrane protein to the amastigote flagellum will be demonstrated to confirm that this protein is involved in flagellar membrane targeting in amastigotes as well as promastigotes. Recently published experiments have demonstrated that the tips of the amastigote flagella form `synapses' with the phagolysomal membrane of the macrophage, suggesting that these synapses could be important in parasite/macrophage interactions. The possibility that Δkharon1 null mutants may fail to form such potentially critical synapses will be tested by electron microscopy. Additionally the possibility that Δkharon1 mutants fail to deliver a protein from the flagellum to the macrophage cytosol will also be examined. These experiments could provide a mechanistic explanation for the avirulence of Δkharon1 null mutants. Finally, preliminary studies have identified several candidate components of the KHARON Complex using biotin proximity labeling. Experiments will address whether these candidate subunits exist in the same high molecular weight complex as KHARON1 in intracellular amastigotes. Overall, this application will elucidate the critical role of the KHARON Complex in parasite virulence.

 描述（应用程序提供）：相关性：寄生原生动物，例如利什曼原虫和锥虫感染，全球数百万人，并引起毁灭性和致命疾病。该应用将阐明这些寄生虫成功侵入人类细胞并引起疾病的重要机制。概括。最近的发现表明，介导这些单细胞寄生虫运动性的类似鞭子样的鞭毛在引起阶段的HE病中起着至关重要的作用，利什曼尼亚的amastigote和Bloodstream形成了非洲锥虫。尽管ΔKharon1null突变体在培养基中复制为轴突膜，但一旦进入宿主巨噬细胞，它们就不会复制，而是死于这些宿主细胞内。这些结果表明，kharon1在巨噬细胞吞噬体内部的杂物生存中起作用。最近的实验还表明，在感染BALB/c小鼠后，ΔKharon1Null突变体是无毒的。 Kharon1是指定Kharon Complex的高分子量多蛋白质复合物的一部分的其他初步数据建立。该应用的总体目的是剖析卡隆复合物在疾病中的关键功能，从而导致amastigotes，从而阐明Amastigote Flagelum在寄生虫病毒中的作用。 kharon1蛋白位于昆虫期前染料中鞭毛轴突的底部，通过高分辨率荧光显微镜和电子显微镜将其定位在amastigotes中。 Kharon1的能力将膜蛋白转移到amastigote Flagelum中，以确认该蛋白与鞭毛有关 膜的膜靶向杂物和前载体。最近发表的实验表明，与巨噬细胞的吞噬性膜体膜形式的木质鞭毛形式的尖端表明这些突触在寄生虫/巨噬细胞相互作用中可能很重要。 Δkharon1无效突变体可能无法形成这种潜在的临界突触的可能性将通过电子显微镜测试。此外，还将检查ΔKharon1突变体无法从鞭毛到巨噬细胞胞质溶胶的蛋白质的可能性。这些实验可以为Δkharon1无效突变体的气相提供机械解释。最后，初步研究已经使用生物素接近标记确定了卡隆复合物的几个候选成分。实验将解决这些候选亚基是否存在于与kharon1相同的高分子量复合物中，在细胞内膜片中。总体而言，该应用将阐明卡隆复合物在寄生虫病毒中的关键作用。